The goal of this project is to define the molecular mechanisms involved in the replication of enveloped RNA viruses and in particular, to understand the factors which influence the regulation and expression of viral genetic information. Studies are being carried out with the murine leukemia virus system. Current interest is focused on the process of reverse transcription in an effort to correlate genetic structure with enzymatic function. A nonconditional viral mutant which is defective in the pol gene has been shown to produce an abnormally small enzyme with reduced polymerase activity, although its genome size is the same as wild type. Recombinant DNA technology is being used to map the genetic lesion in this mutant and a molecular clone of the entire viral genome has now been obtained. Recent experiments involving cotransfection of mammalian cells with combinations of mutant and wild type DNA subclones have made it possible to localize the mutant defect to a 400 base pair region in the middle of the pol gene. Enzymatic studies on endogenous MuLV reverse transcription have also continued. A phenomenon not previously described for this reaction, which we refer to as "enzyme pausing", has been observed. Thus, in addition to full-length viral DNA, the products of the wild type reaction include a large number of minus-strand intermediates, some of which are clearly more prominent than others. The mutant polymerase makes only small intermediates (no larger than 2.5 kb), terminating DNA synthesis near a major pause site. Using published DNA sequence data for wild type MuLV, computer analysis of intermediate bands has been undertaken to identify structural features which contribute to pause signals.